Protection of human corneal endothelial cells

ABSTRACT

The cells of the coverings and linings of internal human and animal tissues, organs and body cavities subject to trauma, such as surgery, may be protected against exfoliation or destruction by the topical application or injection of effective amounts of chondroitin sulfate prior to or during the trauma. Preferably, the chondroitin sulfate is used in physiological solutions in concentrations of about 40 to 55 weight percent, and may be used as a surgical irrigating solution. Intraarticular injection of such chondroitin sulfate solutions into human and animal joints having degenerative joint conditions protects the joint cells, reduces aseptic inflammation and/or prevents further degeneration of cartilage tissue. Solutions for preserving human and animal cells and tissues in vitro for later in vivo use have extended storage life when chondroitin sulfate is added to such storage solutions in effective amounts such as about 1 to 20 weight percent of the storage solution.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of our copending U.S. applicationSer. No. 07/349,987, filed May 8, 1989, now abandoned, which in turn wasa continuation of application Ser. No. 06/677,130, filed Dec. 3, 1984,now abandoned, which in turn was a continuation-in-part of applicationSer. No. 06/239,791, filed Mar. 2, 1981, now U.S. Pat. No. 4,486,416.This application is also related to application Ser. No. 07/162,940.filed Mar. 2, 1988, now U.S. Pat. No. 4,971,955, which was a division ofapplication No. 06/677,130.

FIELD OF THE INVENTION

This invention relates to a method of protecting both human and animalcells which are subject to exposure to trauma. A particularly importantembodiment of this invention concerns using chondroitin sulfate toprotect cells in anticipation of surgical trauma.

BACKGROUND OF THE INVENTION

The therapeutic qualities of hyaluronic acid to aid wound healing havebeen previously reported by E. A. Balasz and D. A. Gibbs in "TheRheological Properties and Biological Function of Hyaluronic Acid",Academic Press (New York), 1970. Ultrapure hyaluronic acid and the usethereof is the subject of U.S. Pat. No. 4,141,973 to E. A. Balazs.Furthermore, it has been shown that sulfated mucopolysaccharides have agreater deturgescence effect on the cornea and that viable cornealstroma incorporate sulphur from a bath containing sulfate. See M. E.Langham, "Macromolecular Organization Of A Connective Tissue", JohnsHopkins Press, 1968; J. A. Capella, H. F. Edelhauser, D. L. Van Horn,"Corneal Preservation", Charles C. Thomas Publisher, 1973.

U.S. Pat. No. 3,211,616 of Zensaku Yosizawa concerns N,O-sulfatedneutral-mucopolysaccharides.

U.S. Pat. No. 1,950,100 of Lathan A. Crandall relates to chondroitincompounds and their preparation. Crandall discloses that chondroitin issuitable for the treatment of such diseases as migraine, urticarialeruptions, peptic ulcers, multiple sclerosis, allergies and hepaticcirrhosis.

It is known that chondroitin sulfates are effective in preventing thedevelopment and evolution of some types of complicated lesions inatherosclerosis. Also the chondroitin sulfates exhibit a marked increaseduring reparative processes which ensue after various injuries.

SUMMARY OF THE INVENTION

There has now been discovered a method to protect both human and animalcell layers and tissues subject to exposure to trauma. This methodinvolves administering a prophylactically effective amount ofchondroitin sulfate to the anticipated site of the trauma (to the cellsand tissues) prior to and/or during exposure to the trauma.Administration of an effective dosage of chondroitin sulfate isparticularly useful to prevent cell damage during surgery and traumasuch as may occur to a joint surface. Additionally, chondroitin sulfatewhich is a known agent for promoting wound healing, can be administeredafter trauma as an aid in healing.

The present invention is particularly directed to protection of thecells of the coverings and linings of all internal body cavities andorgans, such as endothelial cells which line the blood vessels and otherorgans, mesothelial cells which line the abdomen and pleural cavitiesand epithelial cells which line the esophagus and mouth. According tothe invention, chondroitin sulfate is particularly useful as anirrigating solution during surgery in order to prevent cell damage andsubsequent adhesion formation.

Chondroitin sulfate may also be added to conventional or special tissuestorage solutions to increase the time during which animal tissues ororgans may be stored or shipped. Such storage solutions having improvedstorage time may contain an effective amount of chondroitin sulfatetogether with the usual metal ions and other nutrients necessary forstorage of animal tissues.

Still further, chondroitin sulfate has been found to be effective inameliorating aseptic joint inflammation in the knees, hips, feet andother joints of animals, particularly four-legged animals such ashorses. The chondroitin sulfate reduces the inflammation of the jointand protects the surfaces and cells of the joint tissues from furtherdamage and deterioration.

DETAILED DESCRIPTION OF THE INVENTION

Animal connective tissues contain a group of closely related acidiccarbohydrate polymers which are located in the extracellular matrix andare collectively known as mucopolysaccharides. Mucopolysaccharides areheteropolysaccharides formed by the chain condensation of a pair ofmonomeric sugar units in an alternating sequence, and as a result, theselarge polymers are invariably built-up from disaccharide repeatingunits. One of the monomers of mucopolysaccharides is always a hexosamine(2-amino-2-deoxyglycose). The basic amino group of the hexosamine isalways present as the neutral acetamino derivative.

Chondroitin sulfate has three isomers (chondroitin sulfate A,chondroitin sulfate B and chondroitin sulfate C) that are characterizedby a sulfate group taking one of three positions in the repeatingdisaccharide unit (N-acetylchondrosin is the repeating unit). Eachrepeating disaccharide unit has one sulfate group. See The Merck Index,monograph 2194, pages 314-315 (10th ed. 1983).

Chondroitin sulfates A and C both contain D-glucuronic acid, 2 amino-2deoxy-D-galactose, acetyl and sulfate residues in equimolar quantities.There is a close similarity in structure between chondroitin sulfates Aand C, demonstrated by the fact that both afford the same disaccharide,chondrosine, in high yields on acidic hydrolysis. These twomucopolysaccharides are distinguished by their optical rotation and bythe solubility of their calcium salts in aqueous ethanol. Chondroitinsulfates A and C structurally differ only in the position of the sulfateester grouping in the hexosamine residue; the sulfate ester occurs atcarbon-4 in the chondroitin sulfate A and carbon-6 in the chondroitinsulfate C. The nature of the hexuronic acid (L-iduronic acid) ofchondroitin sulfate B serves to distinguish it from other isomericchondroitin sulfates.

The similarity of the carbon skeleton in chondroitin sulfate B to thatof chondroitin sulfate A and C is evident. In all three acidmucopolysaccharides the acid linkage is BI-3, the hexosaminidic linkageis BI-4 and the hexosamine is galactosamine.

The assignment of sulfate ester groups in chondroitin sulfates A and Cto carbon-4 and carbon-6 respectively was initally based upon infraredspectral analysis, since equatorially and axially-located sulfate groupsabsorb at different wave lengths in the region 700-1000 cm⁻¹.Methylation studies have confirmed this assignment in chondroitinsulfate A.

Chondroitin sulfate B is not degraded by pneumoccal or testicularhyaluronidases and can, therefore, be distinguished from hyaluronic acidand chondroitin sulfates A and C because of this action. Its negativeoptical rotation (αD=-55 degrees to -63 degrees) is much greater thanthat of chondroitin sulfate A (α_(D) =-28 degrees to -33 degrees) orchondroitin sulfate C (α_(D) =-16 degrees to -22 degrees).

Chondroitin sulfate is a sulfate acid mucopolysaccharide, which is anormal component of human cornea, and is ubiquitous in animal tissue. Itis a viscous substance that has a molecular weight of about 50,000 to100,000 depending on the source, although lower molecular weight specieson the order of about 20,000 to 50,000 are also sometimes present.Chondroitin sulfate is effective in the present invention at allmolecular weights.

The sulfate ester content of the chondroitin sulfates is subject to widevariations depending on the source. The sulfate ester content ofchondroitin sulfates in corneal extracts is usually consistently low. Incontrast, the sulfate ester content of chondroitin sulfates obtainedfrom shark connective tissues is high when compared to the sulfate estercontent of chondroitin sulfates derived from equivalent mammaliansources. The degree of sulphation is not believed to have anysignificance in the applicability of chondroitin sulfate in the presentinvention.

Mucopolysaccharides are extracellular components having one or twonegatively charged groups per disaccharide repeating unit. Being asulfated mucopolysaccharide, chondroitin sulfate carries an extranegative charge per repeating unit. Chondroitin sulfate has a -2 chargeper unit (a carboxyl group and a sulfate group each supply one negativecharge). The biological macromolecule of chondroitin sulfate forms arandom coiled polymer, with the repulsive forces of the negative chargesmaintaining a minimum volume per molecule. The polyanion itself bindsmany water molecules so that it forms a mucopolysaccharide watercomposite and generates a protective cushion.

An intraocular lens, which is manufactured from a highly electricalinsulating material, strongly attracts the endothelial layer of thecornea when direct tactile contact is made. There is, in fact, an actualtransfer of the endothelial cellular network, as if there were anattraction of the layer by the lens. The highly electrically conductivechondroitin sulfate arrangement is eminently suitable to eliminate thiselectrical attractive force. The extra negative charge associated withchondroitin sulfate also endows it with greater molecular elasticity,making it an excellent lubricating material.

Since acid mucopolysaccharides are viscous, highly hydrated polyanions,showing a marked degree of interaction even in dilute solution, theyimpede the direct flow of fluids and thus contribute to the mechanicalresistance to compression. The mucopolysaccharide will limit the flow ofinflammatory products, for example, proteins.

Certain bacteria, for example, staphylococcus, contain hyaluronidase. Aninfection may spread by depolymerization, i.e. attack upon themucopolysaccharide. Since chondroitin sulfate B does not respond to thisenzyme, the use of chondroitin sulfate B would limit, or even eliminate,the activities of accidental infection during surgical procedures.Chondroitin sulfate also limits the movement of proteins and largemolecules, thus aiding in the control of infection.

Chondroitin sulfate can play an active as well as a passive role. Thus,it can be utilized before, during and after trauma. As a passiveinstrument, chondroitin sulfate physically separates tissues and acts asa lubricant.

In an active role, chondroitin sulfate behaves as a molecular sieve. Itcollects large molecules that participate in adhesion formation andpermits the smaller molecules to permeate through it. Thus there will bean exclusion of large molecules and a space exists along eachchondroitin sulfate aggregate into which no protein can move. Thesieving effect acts as a hindrance to the flow of various types ofmolecules based on the possible entanglement of the polysaccharidechains. This leads to the development of a three dimensional network.Thus large molecules are retarded in relation to their shape, electricalcharge, polar groups, etc. In certain cases chondroitin sulfate sievingcan be augmented by the addition of Ca²⁺ ions.

Chondroitin sulfate itself is broken down as part of the healingprocess. The sulphur atom of chondroitin sulfate can be radioactivelytraced during its incorporation into healing tissue. Chondroitin sulfatediminishes tissue rejection in tissue implants, thus acting in an activerole that serves various surgical procedures including those inophthalmology.

Although both chondroitin sulfate and "HEALON", i.e., high molecularweight (polymerized) fraction of sodium hyaluronate availablecommercially from Pharmacia Labs, Inc., are mucopolysaccharides, thesetwo substances are quite different. "HEALON" has a much higher molecularweight than chondroitin sulfate (the molecular weight of "HEALON" isgreater than 1,000,000 as compared to about 50,000 to 100,000 forchondroitin sulfate). Chondroitin sulfate does not attain the greatsingle chain lengths of "HEALON" molecules. Chondroitin sulfate has onlyabout 50 repeating disaccharide units per chain as compated to more thanabout 15,000 units per chain for "HEALON".

After ophthalmic surgery, "HEALON" remains in the anterior chamber for avery long period of time, i.e., several days. Chondroitin sulfate,however, is completely dissipated after surgery within 24 to 36 hours.

The present invention involves the utilization of chondroitin sulfate asa protective agent prior to and/or during anticipated trauma, such assurgery, to minimize or eliminate cell damage. Chondroitin sulfate mayalso be administered during surgery to foster separation of tissueplanes during surgical procedures including but not limited to generalsurgery, neurosurgery, orthopedic surgery, vascular surgery andgynecological surgery in protecting cells and maintaining body cavitiesand joint cavities and preventing adhesion formations.

The damage to cells during such surgery is generally of twokinds--mechanical and electrostatic. An exemplary protective coating foruse during surgery would thus exhibit the following attributes: theability to absorb the impact of mechanical contact, the ability to actas a good electrical conductor (to eliminate or reduce the electrostaticinteraction between surgical instruments and the cells), high viscosityand biological compatibility. Chondroitin sulfate meets all thesecriteria.

All three isomer forms of chondroitin sulfate (A, B, and C) can beutilized in the present invention. Chondroitin sulfate A can be derivedfrom whale cartilage; chondroitin sulfate B can be derived from porcineskin; and chondroitin sulfate C can be derived from shark cartilage. Afairly plentiful source for chondroitin sulfate is the nasal septa ofcows. Chondroitin sulfate also exists in the trachea, aorta, tendons andother parts of the animal body. Certain sections of domesticated animalcartilage contain up to 40% chondroitin sulfate by weight. All forms ofchondroitin sulfate with varying sulphur contents essentially have thesame mechanism of protective action and would therefore be effective inthe present invention.

Chondroitin sulfate isolated from shark cartilage, and designated aschondroitin sulfate D, has revealed an infrared spectrum identical withthat of chondroitin sulfate C. However, the non-identity of the twopolysaccharides as acceptors in enzymatic sulphation, and the unusuallyhigh sulphur content of chondroitin sulfate D (S, 7.6%; corresponding to1.3 sulfate residues per hexosamine) suggests that they might bedistinct species. Chondroitin sulfates from shark preparations manifestexcellent properties in anterior segment surgery.

Chondroitin sulfate may be utilized in the present invention in variousforms and concentrations depending upon the particular tissue area andmethod of application. One of ordinary skill in the art can readilydetermine with a minimum amount of experimentation the appropriateamount of chondroitin sulfate to be applied depending on such factors asdesired viscosity or coating thickness, whether the chondroitin sulfateis to be applied topically or injected, etc. The concentration ofchondroitin sulfate to be used in solutions depends also upon whetherthe chondroitin sulfate is to be used in vivo as a protective agent orin vitro as a storage and preserving solution.

In the case of in vivo protective solutions, it is preferred that thechondroitin sulfate be present as a component in the physiologicalsolutions in amounts of about 40 to 55 weight percent of the solution,and more preferably about 50 to 55 weight percent of the solution.Concentrations below about 40% by weight may still be satisfactory, butnot as effective as the preferred concentrations indicated above. Forexample, as described in U.S. Pat. No. 4,486,419 a 20 weight percentsolution of chondroitin sulfate was satisfactory for in vivo cataractextractions with rabbits and monkeys. Concentrations above about 60% mayalso be effective, but are not preferred since higher concentrationswill begin to dehydrate tissues to which the chondroitin sulfate isapplied.

In the case of in vitro preservative solutions, concentrations of aboutone to twenty weight percent of chondroitin sulfate are preferred, andmore particularly about one to five weight percent chondroitin sulfate.

The solutions are preferably aqueous solutions which in addition tochondroitin sulfate may be substantially composed of normal salinesolution, basic salt solution or other buffered solutions which arecommonly known and used in handling biological materials. Some of thesephysiological solution bases and other additives are exemplified morefully below for particular applications, and others will be apparent tothose of ordinary skill in the art. A particularly preferred solutionfor use in in vivo applications of the present invention comprises 50%by weight (400mg/ml) chondroitin sulfate in a disodium phosphate(14mg/ml)/monosodium phosphate hydrate (0.13mg/ml) buffer.

The chondroitin sulfate solution can be introduced to the anticipatedarea of the surgery or treatment area by any convenient means such asusing a cannula or needle, i.e., injected into a body cavity, joint orvessel, or using irrigating devices or swabs for topical application.

Protection of Cells and Tissues of Internal Body Tissues, Organs andCavities

During any invasive procedure, such as surgery, the cells of thecoverings and linings of various body organs, tissues and cavities aresubject to trauma from the surgical cutting as well as othermanipulations which may be performed during the invasive procedure.These body linings and coverings are covered with endothelial,mesothelial or epithelial cells. The protection of endothelial cells ofthe cornea has already been discussed in some detail in our U.S. Pat.No. 4,486,416. Endothelial cells also cover the inside of other bodyorgans such as blood vessels. Mesothelial cells are the flat squamouscells which cover the intestines and the true serous membranes of thebody cavity including the peritoneum, the pleural cavity and thepericardium. While epithelial cells are primarily found covering theoutside surfaces of the body, they also line the esophagus and theinside of the mouth.

The use of chondroitin sulfate to protect cells which cover the internalbody coverings and linings according to the present invention isparticularly advantageous during invasive procedures such as surgery,and may be used in irrigating solutions in place of or as an additive toconventional irrigating solutions such as normal saline or Ringer'ssolution to flush tissue surfaces during surgery. The prophylacticeffect of chondroitin sulfate in abdominal surgical procedures isdemonstrated by the following experiments:

Sprague Dawley white rats were anesthetized intraperitoneally withsodium pentobarbital, and a celiotomy was performed by removing a stripof skin using scissors and similarly opening the linea alba. A loop ofintestine from the rats was delivered onto a polypropylene apron whichwas attached to the belly of the rats with towel clamps and moistenedwith buffered mammalian Ringer's solutions (BRS). The loop of intestinewas also moistened with BRS and kept moist throughout the experiment.Care was taken that the portion of the gut being investigated was not apart actually contacted by fingers or surgical instruments. At the endof an experiment each rat was sacrificed by intracardiac injection ofstrong pentobarbital solution.

A series of experiments was performed on the exposed loop of intestineof each rat with washing, abrading, staining and application ofchondroitin sulfate in various orders of performance, with one or moreof these steps being omitted for comparison. The abrasion was done witha Halda force gauge with a curved wire tip. To the extent possible, anabrasion consisted of 15 strokes of the tip against the side of theintestine throughout a longitudinal distance of 1 cm such that theneedle of the gauge remained between 10 and 15 grams for 90% of eachstroke. The abrasion was applied along the long axis of the intestine,and the mesentery was not abraded due to the tendency of the stainingdye to leak under the loop of gut so as to be inaccessable to washingand give the false appearance of staining. The staining consisted ofapplying at least 1 ml of the purple stain, Acid Violet TFH (more wasapplied if the first application did not cover the area), and the stainwas allowed to remain for at least twenty seconds and not more thanabout 45 seconds. Washing or flushing was done by copious rinsing withBRS (at least 25 ml per site from the open end of an intravenousinfusion tube). In final washes after staining, enough BRS was useduntil the fluid .pa draining away from the area of staining wasuncolored to visual examination (usually 25-100 ml but as much as 300ml).

The chondroitin sulfate solution used was a 50 weight percentchondroitin sulfate solution in phosphate buffer as described above. Inthe results described below "random staining" refers to the appearanceof areas that took the stain (generally very lightly) in a mannerunrelated to the experimental manipulations and with no recognizableborders or edges to the stained part. This may be due to handling and/ordrying of the tissues. "Distinct staining" refers to areas havingrecognizable edges and rather uniform depth of staining. Five series ofexperiments with five or six rats each were performed as described belowwith the indicated results:

Series No. 1 (Average weights of rats=140 gm)

1. One ml CDS intraperitoneally before celiotomy; washed copiously afteropening; abraded; stained; washed. Visual appearance: deep distinctstaining of abraded area and especially mesenteric area.

2. One ml CDS intraperitoneally before celiotomy; abraded; stained;washed copiously (did not wash after opening). Visual appearance: verylightly stained.

3. One ml CDS intraperitoneally. Died.

4. CDS locally after opening and washing; no abrasion; stained; washed.Visual appearance: very little and very light staining.

5. No CDS; opened; washed; abraded; stained; washed. Visual appearance:deep discrete staining at place of abrasion; some light "randomstaining".

Series No. 2 (Average weight of rats =165 gm)

1. One ml CDS intraperitoneally; wash; no abrasion; stain; wash. Visualappearance: no staining.

2. One ml CDS intraperitoneally; abrade; flush; stain; flush. Visualappearance: no staining.

3. One ml CDS intraperitoneally; flush first; abrade; flush; stain;flush. Visual appearance: distinct staining.

4. Two drops CDS locally after opening on left; abrade on both sides;flush; stain; flush. Visual appearance: CDS part much less staining thannon-CDS, some "random" staining.

5. Two drops CDS locally on right after abrading; flush; stain; flush.Visual appearance: both sides stained, some "random" staining.

Series No. 3 (Average weight of rats =200 gm)

1. Flushed; Two drops of CDS on left; abraded both sides; washed;stained; washed. Visual appearance: slight stain on left (CDS), no stainon right (no CDS).

2. Flushed; Two drops of CDS on left; abraded both sides; washed;stained; washed. Visual appearance: no stain on left (CDS), stained onright (no CDS).

3. No flush on opening; Two drops CDS on right; abraded both sides;washed; stained; washed. Visual appearance: light stain on right (CDS),heavy distinct stain on left (no CDS).

4. Flush; Two drops CDS locally on right; abrade both sides; wash;stain; wash. Visual appearance: right side (CDS) no stain, left side (noCDS) heavy distinct stain.

5. Woke up.

Series No. 4 (Average weight of rats =250 gm)

All had sequence of: wash; apply CDS (two drops) locally to one part;abrade both sides; wash; stain; wash.

1. CDS on right. Visual appearance: some random staining, CDS part muchless than non CDS part.

2. CDS on left. Visual appearance: left side (CDS) no stain, right side(no CDS) deep stain.

3. CDS on right. Visual appearance: right side (CDS) no stain, left side(no CDS) moderate but distinct staining.

4. CDS on left. Visual appearance: left side (CDS) no stain, right side(no CDS) deep staining.

5. Woke up, given more pentobarbital but still awake, died later.

Series No. 5 (Average weight of rats=250 gm)

1. (Control) wash; (no abrasion, no CDS) stain; wash. Visual appearance:slight random staining.

2. Wash; abrasion on left only; (no CDS) wash; stain; wash; Visualappearance: clear distinct staining of abraded part, light randomstaining.

3. (No wash) Two drops CDS locally on right; wash; stain. Visualappearance: random staining on CDS part, distinct staining on non CDSpart.

4. Wash, Two drops CDS locally on left; abrade both sides; wash; stain;wash. Visual appearance: left side (CDS) light stain, right side (noCDS) heavy distinct stain.

5. Wash; two drops CDS locally on right; abrade both; wash; stain; wash.Visual appearance: right side (CDS) random staining, left side (no CDS)distinct staining.

6. Wash; two drops CDS locally on left; abrade left (CDS part only);wash; stain; wash. Visual appearance: left side (CDS) no stain, rightside (no CDS, no abrasion) random staining.

Application of the chondroitin sulfate (CDS) preparation either locallyor intraperitoneally, before abrasion of the visceral mesentericsurface, in all sixteen rats tested prevented or significantly decreasedthe staining of abraded areas by the Acid Violet dye, thus supportingthe hypothesis that CDS prevents or moderates the exfoliation ordestruction of cells from the visceral mesenteric surface.

Chondroitin Sulphate in Storage Solutions

Various media have been prepared to successfully preserve animal tissuesfor storage and/or transportation for later use. However, even with suchspecially prepared storage media, the practical, useful storage life islimited to a few days. For example, MK medium (See McCarey, BE andKaufman, HWE, "Improved Corneal Storage", Investigational Ophthalmology13:165 (1974) has been shown to successfully preserve corneal tissue ofrabbits for as long as fourteen days but subsequent studies (seeKaufman, H. E. et al. "Chondroitin Sulfate in a New Cornea PreservationMedium", American Journal of Ophthalmology, 98:112 (1984)) indicatedthat inpractice the MK medium could preserve corneal tissue in storageonly for up to four days, whereas it would be desirable to be able tostore corneal tissue in a refrigerator with no additional processing forat least two weeks.

It has been found that the addition of chondroitin sulfate in amounts ofabout 1 to 20, and preferably about 1 to 5 weight percent toconventional or special storage media will significantly extend thestorage capabilities of the media up to several weeks. A particularlypreferred aqueous solution contains 2.5% CDS in 0.02M HEPES, TC 199 forosmolarity, sodium bicarbonate and gentamicin sulfate. It will beunderstood that chondroitin sulfate alone, or a buffered solution ofchondroitin sulfate alone will not suffice as a storage media, since itis necessary to include the metal ions and other nutrients needed by theviable tissue. The ability of chondroitin sulfate to extend the storagecapacity of animal tissues in storage media is illustrated by thefollowing experiments performed following our suggestions:

The solutions which were used as test media with or without chondroitinsulfate (CDS) included BSS (basic salt solution), BSS Plus (acommercially available long term (1-6 hours) irrigating solution), MEM(minimal essential medium), TC 199 (a standard commercially availabletissue culture medium), HEPES (a standard commercially available cellculture buffer system), FLUOSOL (a blood substitute available from TheGreen Cross Corporation), AMVISC (a 1% sodium hyaluronate in BSScommercially available from Medchem and Precision Cosmet), Hanks BSS (acommercially available tissue culture medium) without phenol redindicator, HEALON, and dextran of different molecular weights. All testsolutions contained 200 ug/ml gentamicin sulfate. Low molecular weightcomponents of chondroitin sulfate were removed for the sake ofuniformity.

Human corneas initially perserved in MK (McCarey-Kaufman) medium for oneto three days or whole globes stored in moist chambers for one to threedays were stored in the experimental media for two weeks. Viability ofthe endothelial cells was assessed morphologically by trypan bluestaining. Some corneas were examined by electron microscopy. The resultsare shown in Table I.

The solutions containing 2.5% to 10% chondroitin sulfate, TC199 andHEPES buffer yielded the most satisfactory results. Grafts stored in amedium containing either 2.5% or 5% chondroitin sulfate displayedminimal trypan blue staining and normal structure by electronmicroscopic anaylsis. Subsequently, such corneas have been used inanimal eyes and in five human eyes with satisfactory results in terms ofclarity.

                                      TABLE I                                     __________________________________________________________________________    Analysis of Corneas and Globes Preserved for 14 Days at Refrigerator          Temperature (4 C.) in Various Media                                                                      Days from    Gross Appearance                                                 Death of Donor to                                                                          After 14-day Endothelial Cells                         No. Tested                                                                              Beginning of Study                                                                         Storage in   Stained With             Composition of Medium                                                                          Globes                                                                            Corneas                                                                             Globes                                                                              Corneas                                                                              Experimental Medium                                                                        Trypan Blue              __________________________________________________________________________                                                         (%)                      TC 199, HEPES, 2.5% CDS                                                                        1   1     2     2      Clear        0 to 1                   TC 199, HEPES, 10% CDS                                                                         1   1     1     4      Clear to slightly                                                                           5oudy                   TC 199, HEPES, 1% CDS                                                                          3   6     2 to 3                                                                              2 to 3 Clear to slightly                                                                          60oudy                   TC 199, HEPES, dextran                                                                         3   5     1 to 3                                                                              2 to 4 Slightly cloudy to                                                                         60oudy                   BSS, glucose, HEPES, 10% CDS                                                                   0   2     --    2 to 3 Clear         0                       BSS, glucose, HEPES, 1% CDS                                                                    2   4     3     1 to 3 Clear to Cloudy                                                                            50                       BSS, glucose, HEPES, dextran                                                                   2   5     2 to 3                                                                              1 to 3 Clear to Cloudy                                                                            60                       TC 199, 20% CDS  4   2     1 to 2                                                                              2 to 3 Clear        0 to 1                   TC 199, 10% CDS  5   3     0.25 to 2                                                                           2 to 3 Clear to slightly                                                                          16oudy                   TC 199, 5% CDS   1   1     2     1      Clear to slightly                                                                          50oudy                   TC 199, 2% CDS   1   1     2     2      Slightly to medium                                                                         50oudy                   BSS, 10% CDS     1   1     3     3 to 4 Cloudy       100                      BSS, 5% CDS      1   2     1     3      Cloudy        0.sup.#                 BSS, 2% CDS      2   1     1 to 3                                                                              3      Cloudy        0.sup.#                 BSS Plus, 20% CDS                                                                              2   0     1 to 2                                                                              --     Clear        ND.sup.+                 BSS Plus, 10% CDS                                                                              2   0     0.25 to 2                                                                           --     Cloudy       50                       BSS Plus, 5% CDS 2   0     1 to 2                                                                              --     Cloudy       45                       BSS Plus, 2% CDS 1   1     2     3      Clear to Cloudy                                                                            95                       TC 199, 0.2% fat-free albumin                                                                  1   2     2     1 to 2 Cloudy to clear                                                                            50                       MEM, 0.2% fat-free albumin                                                                     1   1     2     1      Clear to slightly                                                                          65oudy                   MEM, 20% CDS     1   0     1     --     --           30                       MEM, 10% CDS     1   1     1     4      Slightly cloudy to                                                                         65oudy                   Fluosol          0   1     --    3      --           30                       TC 199, 50% Amvisc                                                                             2   0     2     --     Slightly cloudy to                                                                         50oudy                   TC 199, 50% Healon                                                                             1   0     2     --     Slightly cloudy to                                                                         50oudy                   TC 199*, 50% Healon                                                                            1   0     2     --     Cloudy        0.sup.#                 M-K medium       1   0     2     --     Slightly cloudy                                                                            100                      Modified M-K medium                                                                            0   2     --    0.33 to 2                                                                            Cloudy to slightly                                                                         50oudy                   __________________________________________________________________________     .sup.# No endothelium.                                                        .sup.+ Not done; tissue used as graft in monkey eye;                          *Double strength.                                                        

One patient who received a graft that had been stored in theexperimental medium for twelve days demonstrated a complete endothelialsheet and an endothelial cell density of 1800 cells/mm two weeks aftersurgery. Patients receiving transplants stored 14 and 16 days also hadnormal post-operative graft thickness.

Chondroitin Sulfate Treatment of Aseptic Inflammation of Animal Joints

Aseptic inflammation (i.e., without infection by microorganisms) of thejoints of animals, particularly four-legged animals such as horses, is aserious characteristic of degenerative joint disease. As the cartilagecells of the joint degenerate, the synovial fluid (which is largelyhyaluronic acid) of the joint is of such inferior quality that it hasvery poor lubricating capacity (i.e., it becomes nonviscous), so thatinflammation of the joint results.

It has been found that the injection of effective amounts of chondroitinsulfate, which is a significant component of the articular cartilage,into the inflamed joint reduces inflammation and prevents furtherdegeneration of the cartilage (mainly through protection of cells in thejoint cavity and also through lubrication and cushioning effects) andpossibly causes some regeneration of cartilage through stimulation ofmucopolysaccharide synthesis by the chondrocytes. That is, thechondroitin sulfate provides a protective effect against furthercartilage damage, with possibly the mechanical and/or hydrodynamicpotentiation of healing or regenerative effects.

Chondroitin sulfate may be injected intraarticularly in generally thesame types of vehicles (physiologic solutions) as described above forsurgery and other in vivo cell protection, preferably at concentrationsof about 40 to 55 weight percent. The amount of solution to be injectedvaries depending upon the particular joint and joint size being treated.As an example, about 4 cc of a 50 weight percent solution issatisfactory for a horse leg joint.

While chondroitin sulfate has been found to be particularly advantageousin the treatment of the hock, knee and fetlock joints of horses, it isbelieved that chondroitin sulfate broader application in treating theleg joints of other animals including those of humans and larger dogs,cattle, etc. having similar joint problems. For example, chondroitinsulfate may be useful in treating arthritic joints and otherdegenerative joint conditions in humans and other animals. Theeffectiveness of chondroitin sulfate in treating equine joints isdemonstrated by the following experiments:

Forty-six standardbred horses with no more than one affected joint at atime were included in the study. None of the horses had fractures andnone had received other intra-articular therapy in the affected jointwithin the previous four weeks. Of the total of 54 joints treated duringthe study, 45 joints were treated with a 50 weight percent chondroitinsulfate solution with phosphate buffer as indicated above (hereinafterreferred to as CDS or the CDS therapy), and 9 joints were treated withthe commercially available "HYLARTIL VET," (an equine product similar to"HEALON" available from Pharmcia Labs, Inc. comprising 1% sodiumhyaluronate).

Prior to treatment, joint temperature and lameness were measured andrecorded. A sample of synovial fluid was removed from the test joint andreplaced with an equal volume of the treatment solution or 4 ml. beingcareful to avoid extensive "overfilling" of the joint. Temperature andlameness were evaluated at one and two weeks after treatment. If thejoint condition was considered to be 50% or less improved, then a secondadministration of the test solution was made equal to the first, andtemperature and lameness were again evaluated weekly.

Analysis of synovial fluid revealed no consistent correlation withclinical results. However, joint temperature, stride and race testresults correlated well with clinical observations. Table II shows themean temperature differences for inflamed joints minus control jointsprior to and after treatment. Joint temperatures prior to treatment wereon the average 3.6 degrees C. higher than those of the opposite controljoint. Overall, the 45 joints treated with CDS showed improvement of74.2% and 85.3% one and two weeks respectively after therapy, while theHYLARTIL VET therapy in 9 joints showed improvement of 71.5% and 82.8%one and two weeks, respectively after therapy.

Since the horse favors the affected limb and places the least amount ofweight possible for the shortest possible time on that limb, thereappears to be a difference between the length of stride made by theanterior limb versus the posterior limb. With severely affected joints,the anterior limb stride is longer than the posterior limb stride, butas the inflammation subsides, the anterior stride shortens and theposterior stride lengthens with modest change in the sum of the stridelengths. The stride lengths of the anterior limb and the posterior limbapproach equality as recovery to soundness is achieved, and the sum ofstride lengths may increase as soundness of all limbs is achieved.

As shown in Table III the stride length decreased for anterior limbs andincreased for posterior limbs after therapy with either CDS or HYLARTILVET. Both treatments resulted in improvement of the stride in the horsefavoring an affected limb, and, in fact, the CDS therapy showed a slightincrease in the sum of the stride length, suggesting return to soundnessof the limbs.

Actual racetrack performances of 25 horses treated with CDS and 5treated with HYLARTIL VET were obtained and compared before and aftertherapy. All but 5 of the CDS treated horses showed improvement inperformance (decrease in elapsed time to complete the race), while 3 ofthe 5 HYLARTIL VET treated horses showed improvement. Table IV shows theperformance data of these horses. CDS treated horses improved bydecreasing their elapsed race time by an average of 2.2 seconds, whilethe HYLARTIL VET treated horses decreased elapsed race time by 0.8seconds.

The results of the foregoing tests correlated well with clinicalevaluations which showed clinical improvement as joint inflammationsubsided and the joint returned to soundness. The results of the studysuggest that CDS therapy is equally effective, if not more so, asHYLARTIL VET for indications of aseptic inflammation in the equinejoint.

                                      TABLE II                                    __________________________________________________________________________    EQUINE JOINT TEMPERATURE                                                      CDS THERAPY             HYLARTIL VET THERAPY                                  JOINT                                                                              Pretherapy                                                                            1 wk 2 wk  Pretherapy                                                                           1 wk 2 wk                                      __________________________________________________________________________    Knee N = 16 3.5 C.                                                                         1.2 C.                                                                             1.1 C.                                                                              N = 5 3.0 C.                                                                         0.6 C.                                                                             0.2 C.                                                 (66%)                                                                              (69%) (80%)       (93%)                                     Front                                                                              N = 27 3.6                                                                            0.7  0.3   N = 2 4.5                                                                            2.0  1.5                                       fetlock      (81%)                                                                              (92%)        (56%)                                                                              (67%)                                     Hind N = 1 0.0                                                                             0.0  0.0   N = 1 9.0                                                                            3.0  2.0                                       Fetlock                        (66%)                                                                              (78%)                                     Hock N = 11 4.0                                                                            0.7  0.0   N = 1 2.0                                                                            0.0  0.0                                                    (83%)                                                                              (100%)       (100%)                                                                             (100%)                                    All  N = 45 3.6                                                                            0.93 0.53  N = 9 3.9                                                                            1.11 0.67                                      Joints       (74.2%)                                                                            (85.3%)      (71.5%)                                                                            (82.8%)                                   SEM =                                                                              +/-0.52 +/-0.25                                                                            +/-0.26                                                                             +/-1.23                                                                              0.39 0.29                                      __________________________________________________________________________     The data of this table represent the mean values of the temperature           difference between the inflamed joint and the control joint.                  N = number of joints evaluated.                                               SEM is the standard error of the mean.                                   

                                      TABLE III                                   __________________________________________________________________________    STRIDE TEST ANALYSIS                                                          (Change in Stride Length -inches- After Therapy)                              INJURED JOINT                                                                           CDS THERAPY        HYLARTIL VET THERAPY                             __________________________________________________________________________    KNEE      ANT. -0.63 +/- 0.51 inches                                                                   N = 5                                                                             ANT. 0.40 +/- 0.60 inches                        N = 16    POST. 1.0 +/- 0.40 POST. 0.00 +/- 0.32                                        SUM 0.37 +/- 0.34  SUM 0.40 +/- 0.68                                FRONT     ANT. -1.94 +/- 0.74                                                                          N = 2                                                                             ANT. -1.0 +/- 1.0                                FETLOCK   POST. 2.06 +/- 0.78                                                                              POST. 1.0 +/- 1.0                                N = 27    SUM 0.12 +/- 0.19  SUM 0.0 +/- 0.0                                  HIND      ANT. 0.0       N = 1                                                                             ANT. -5.0 +/- 0.0                                FETLOCK   POST. 1.0          POST. 5.0 +/- 0.0                                N = 11    SUM 1.0 +/- 1.0    SUM 0.0 +/- 0.0                                  HOCK      ANT. -3.0 +/- 1.10                                                                           N = 1                                                                             ANT. -1.0 +/- 1.0                                N = 1     POST. 3.0 +/- 1.10 POST. 0.0 +/- 0.0                                          SUM 0.0 +/- 0.47   SUM -1.0 +/- 1.0                                 __________________________________________________________________________

                  TABLE IV                                                        ______________________________________                                        IMPROVEMENT OF RACETRACK PERFORMANCE                                          CDS THERAPY           HYLARTIL VET THERAPY                                           RACE TIME                RACE TIME                                     HORSE  CHANGE (SEC)   HORSE     CHANGE (SEC)                                  ______________________________________                                        AU     -5             AS        0                                             BN     -2             KLM       +1                                            BA     -3             MBC       -2                                            BL     -1             MBF       0                                             CM     -1             WD        -3                                            CL     -2             N = 5 M = -0.8                                          CR     -2             SD = 1.47                                               CS     -4             SEM = 0.74                                              FL     -3                                                                     CGA    0                                                                      GS     -1                                                                     GS     -1                                                                     GS     -2                                                                     LD     -4                                                                     MEN    -4                                                                     NN     +2                                                                     PN     -5                                                                     PP     0                                                                      RW     -2                                                                     SS     -3                                                                     SD     -2                                                                     SB     +1                                                                     TO     -5                                                                     TTB    +4                                                                     TTB    -10                                                                    N = 25 M = -2.2                                                               SD = 2.67                                                                     SEM = 0.55                                                                    ______________________________________                                         The values in this table are drived from registered track records of each     horse. The change in time represents the time of succeeding races after       treatment minus the time of the race preceeding therapy. All times were       registered within one to two weeks prior to therapy and three to four         weeks after therapy.                                                     

It will be recognized by those skilled in the art that changes may bemade to the above-described embodiments of the invention withoutdeparting from the broad inventive concepts thereof. It is understood,therefore, that this invention is not limited to the particularembodiments disclosed, but it is intended to cover all modificationswhich are within the scope and spirit of the invention as defined by theappended claims.

We claim:
 1. A method for the in vitro storage and preservation of theviability of human corneal endothelial cells for later use, comprisingthe steps of:(a) removing the cornea from the human eye globe; (b)storing the cornea in a solution providing metal ions and cellnutrients, said solution comprising a tissue culture medium, a buffersystem for the solution, an antibiotic, and about 2.5 to 20 weightpercent of chondroitin sulfate; wherein the viability of said cells ismaintained in said solution for a period of greater than 4 days up to atleast about 2 weeks.
 2. A method according to claim 1, wherein saidtissue culture medium comprises TC
 199. 3. A method according to claim1, wherein said buffer system comprises HEPES.
 4. A method according toclaim 1, wherein said antibiotic comprises gentamicin.
 5. A methodaccording to claim 1, wherein said solution consists essentially ofHEPES, TC 199, an antibiotic, and about 2.5 to 20 weight percent ofchondroitin sulfate.
 6. A method according to claim 5, wherein saidantibiotic comprises gentamicin.
 7. A method according to claim 5,wherein said solution comprises about 2.5 to 10 weight percent ofchondroitin sulfate.
 8. A method according to claim 1, wherein saidlater use is in vivo use.
 9. A method according to claim 8, wherein saidin vivo use is a graft or transplant.
 10. A method according to claim 1,wherein said solution comprises about 2.5 to 10 weight percent ofchondroitin sulfate.